Process for extracting pure fractions of lactoperoxidase and lactoferrin from milk serum

ABSTRACT

A process for extracting pure fractions of lactoperoxidase and lactoferrin from milk serum is described. The milk serum is microfiltered and passed through a strong cation exchanger at a high rate of flow for selective adsorption of lactoperoxidase and lactoferrin, and then the lactoperoxidase and lactoferrin are eluted successively and selectively with saline solutions having different concentrations.

The present invention relates to a process for extracting pure fractionsof lactoperoxidase and lactoferrin from milk serum. By milk serum ismeant both skim milk and whey.

In cheese-making, a large amount of whey is obtained as a by-product.Whey has a dry solids content of about 6%, which is composedapproximately as follows:

    ______________________________________                                                         % by weight                                                  ______________________________________                                        Lactose            4.6                                                        Protein            0.6 thereof                                                Lactoperoxidase    0.0020                                                     Lactoferrin        0.0030                                                     Fat                0.05 (after separation)                                    Salts              0.7                                                        Dry solids content about                                                                         6.0                                                        ______________________________________                                    

The protein fraction which constitutes about 10-12% of the dry solidscontent is composed of a number of different protein components Thebiggest are β-lactoglobulin, α-lactalbumin and bovine serum-albumin.Also a number of bioactive components belong to the protein fraction,for example immunoglobulins, lactoperoxidase, lactoferrin and lysozyme.

Both lactoperoxidase and lactoferrin have antimicrobial properties.There is a great interest in extracting natural antimicrobial substancesto be used in new contexts in food technology and in thechemico-technical and medical fields.

There are low contents of these substances in skim milk and whey (alsoin the original milk). Lactoperoxidase and lactoferrin are present incontents of 15-50 mg/litre, depending on the lactation state of the cow.Large quantities of whey (milk) must thus be filtered to facilitateextration of kilogram amounts of these bioactive components.

The process engineering conditions for isolating lactoperoxidase andlactoferrin, respectively, from milk/whey are based on the fact that theisoelectric point (pI) for these two proteins is about 9.5, while themain part of the whey proteins have isoelectric points around 5.1-5.4and the casein at about 4.6. A fundamentally suitable process forseparation of lactoperoxidase and lactoferrin is therefore to contactthe milk/whey with a cation exchanger at a pH of <6 for selectiveadsorption, and use is here made of the positive net charge oflactoperoxidase and lactoferrin, which distinguishes from that of othermilk proteins which have a negative charge at this pH.

The traditional way of isolating lactoperoxidase and lactoferrin insmall amounts for the purpose of research is to use the precipitationtechnique and ion exchange chromatography, frequently combined with gelfiltration, see Morrison, M., Hamilton, H-B., Stotz, E., J. Biol. Chem.228:767 (1957); and Morrison, M., Hultquist, P-E., J. Biol. Chem.238-2847 (1963). These methods are not suited for preparing largeamounts of the bioactive components at issue in an economicallydefensible manner.

U.S. Pat. No. 4,436,658 (Pevrosuset) discloses adsorption of lactoferrinfrom casein-free milk serum (whey) by means of a silica column. The pHof the milk serum is adjusted to 7.7-8.2 before adsorption on thecolumn. Immunoglobulins, lactoferrin and lactoperoxidase adhere to thecolumn. After the adsorption phase, elution with a diluted salinesolution at a pH of <4 takes place. No selective elution of the adsorbedproteins is obtained, particularly not regarding lactoperoxidase. Acolumn holding about 5 g of silica compound can treat 1 litre of whey.This prior art process must be regarded as unsuitable for application onan industrial scale.

Zagulski et al. in Prace in Materialy Zootechniczne 20, (1979), p.87-103 describes a batchwise method of obtaining lactoferrin, in whichuse is made of a weak cation exchanger which is mixed with milk. Afterequilibration, the ion exchanger is applied to a column for elution ofthe adsorbed proteins with a saline solution. The method thus is basedon a batchwise process, and a further purification must be carried outin a second ion exchange step to obtain a high purity of thelactoferrin.

A similar process is described in BE patent specification 901,672 (J. P.Prieels and R. Peipper, Oleofina S.A.). Here, use is made of an ionexchanger based on calcium alginate, in which the ion exchangefunctionality has been obtained by admixture of oxides of zirconium,titanium, quartz or aluminium. The milk/ whey is contacted with the ionexchanger in a packed column or by mixing in a tank, whereby proteinshaving an isoelectric point above 7.5 are adsorbed. After equilibration,the gel is separated mechanically and supplied to a means for washingand eluting with a calcium chloride solution. All fluids contacting thecalcium alginate gel must contain at least 0.1% CaCl₂ to prevent the gelfrom being dissolved. No fractionating of lactoperoxidase andlactoferrin is obtained in the elution, but the fractionating must becarried out in a separate purification step.

As a reason for not working with a commercially established ion exchangetechnique in a column process, the above-mentioned BE patentspecification mentions the unsurmountable difficulties of clogging ofthe ion exchanger caused by the occurrence of particles of globular fatand protein aggregate in the medium.

GB patent specification 2,179,947 discloses a process for the extractionof lactotransferrin from milk. The process is carried out such that thewhey is subjected to ultrafiltration, whereby the protein content of thewhey (including the lactotransferrin) is concentrated about 5 times,whereupon its pH and ionic strength are adjusted. The milk serum thustreated is passed at a very low rate (about 0.03 bed volumes per minute)through an ion exchange column, preferably a weak cation exchanger. Thecolumn is eluted, still at a low rate, with a solution having an ionicstrength gradient which increases up to 0.4 M, when the lactotransferrinis eluted. This is a small-scale process which is not suitable forindustrial preparation of lactoferrin. The use of a weak cationexchanger results in a poor capacity. Whey in an amount of 100 bedvolumes, converted into a natural dry solids content, can pass throughthe ion exchange column between each elution. The problem of clogging ofthe ion exchange filter caused by fat and protein particles has not beensolved by this prior art process.

The following requirements can be placed on an industrially applicableprocess for economic recovery of lactoperoxidase and lactoferrin fromwhey/skim milk:

1) High selective capacity of the adsorption mass. Since the contents oflactoperoxidase/lactoferrin are low in milk serum, the volumes of milkserum which can be treated in one elution, must be large.

2) High rate of flow in the adsorption phase. (Normal chromatographicprocesses usually work at low rates, 0.01-0.10 bed volumes per minute.The reason is that owing to the small particle size, the bed usuallygives high pressure drops, and that the reaction kinetics for theadsorption process frequently require a high rate of flow.

3) The process must be hygienic, which means that the adsorption massmust stand at least a lye treatment at pH 13-14.

The object of the present invention is to achieve a process thatsatisfies the above-mentioned requirements for extraction of purefractions of lactoperoxidase and lactoferrin from milk serum (whey) on alarge scale and at a low cost.

The present invention relates to a process for extracting pure fractionsof lactoperoxidase and lactoferrin from milk serum, said process beingcharacterised by microfiltering the milk serum, passing it through astrong cation exchanger at a high rate of flow for selective adsorptionof lactoperoxidase and lactoferrin, and successively eluting thelactoperoxidase and the lactoferrin selectively with saline solutionshaving different concentrations.

According to the invention, a process is provided for preparing purefractions of two different serum proteins in a single ion exchange step.This has not previously been achieved on an industrial scale. Prior artmethods for extracting these proteins on an industrial scale haverequired two or three purification steps.

The above-mentioned problems of clogging of the ion exchanger, which iscaused by the occurrence of particles, such as globular fat and proteinaggregates, in the serum or whey, are solved according the invention inthat the milk serum (whey) is microfiltered, for example in a so-calledcross-flow process, before contacting the ion exchange bed. By choosinga suitable pore size of the microfilter, fat and protein aggregateparticles which cause clogging, can be eliminated. A suitablemicrofilter has a pore diameter of 0.10-2 μm, preferably 0.4-1.5 μm.

As the starting material for the process according to the invention,milk serum (whey) is used, i.e. milk freed from fat and casein. The milkserum is first treated by microfiltration for removal of residues of fatand protein aggregate particles, preferably in a so-called cross-flowprocess. The microfiltered milk serum is then passed at a high rate(about 1-1.5 bed volumes per minute) through a column packed with astrong cation exchanger which selectively adsorbs lactoperoxidase andlactoferrin. This cation exchanger has excellent rate and adsorptionkinetic properties and a capacity of about 1000 bed volumes of milkserum. This means that about 1000 bed volumes of milk serum can passbefore the lactoperoxidase which has the weakest bond, breaks through,i.e. the ion exchange mass is saturated with these proteins. Merely aslight increase of the pressure drop occurs between the beginning andthe end of the adsorption phase.

The elution of the ion exchange mass is started by washing the milkserum out of the column with a buffer, preferably a phosphate buffer atthe pH of the milk serum, 6.5. Subsequently, impurities, if any, areeluted with a buffer solution containing a weak saline solution,preferably of an inorganic alkali, alkaline earth or ammonium salt, forexample 0.075 M NaCl.

After this preparatory elution, the desired proteins are selectivelyeluted with buffer solutions containing saline solutions selected fromthe above-mentioned salts, at different concentrations. Thus, theelution of lactoperoxidase is performed at a salt concentration in therange of 0.10-0.4 M, and of lactoferrin at a salt concentration within0.5-2 M.

After this treatment, the proteins concerned have been concentratedabout 500 times.

The pure protein fractions are collected, and then a furtherconcentration is preferably effected by ultrafiltration followed bydesalination and freeze-drying so as to obtain a commercial productconsisting of about 90% pure protein fractions.

For the production of 1 kg lactoperoxidase and 1 kg lactoferrin, about65 and, respectively, 45 m³ of whey are required. The purity of theextracted components exceeds 90%. This is obtained by a suitable choiceof ion exchanger and a careful choice of adsorption and elutionconditions in which the pH and the salt concentrations are importantparameters.

The invention will now be described in detail by means of the Examplebelow and the accompanying drawings.

FIG. 1 is a schematic view of a preferred embodiment of the processaccording to the invention;

FIG. 2 illustrates the UV absorption spectrum when elutinglactoperoxidase and lactoferrin from an ion exchange column; and

FIGS. 3a and 3b are chromatograms showing the purity of lactoperoxidaseand lactoferrin after the fractionating according to the invention hasbeen carried out.

EXAMPLE

100 litres of pasteurised and sludge-centrifuged sweet whey at pH 6.5were microfiltered in a cross-flow process at 50° C. By themicrofiltration, remaining residues of globular fat were removedtogether with occurring protein aggregates. The pore size of themicrofilter was 1.4 μm.

After cooling, the whey was passed through an ion exchange column packedwith 80 ml of a specially treated strong cation exchanger (S-Sepharose,fast flow, Pharmacia) on an agarose basis. The height of the bed wasabout 4.1 cm, and the rate through the column was 100 ml/minute,corresponding to a rate of 1.25 bed volumes per minute. The pressuredrop before the column at the beginning of the run was 0.26 bar. 15 hlater, the rate was still 100 ml/minute at a pressure drop of 0.28 bar.The lactoperoxidase break-through occurred when about 80-90 litres ofwhey had been passed through the column, i.e. about 1000 bed volumes.

Subsequently, the flow of whey was interrupted, and the eluting phasewas started by washing the whey out of the column with a phosphatebuffer, 0.01 M KH₂ PO₄, pH 6.5, followed by elution of impurities fromthe ion exchanger with a phosphate buffer containing 0.075 M NaCl (FIG.1). The lactoperoxidase was eluted with a phosphate buffer containing0.3 M NaCl, and then the lactoferrin was eluted with a phosphate buffercontaining 0.9 M NaCl (see FIG. 2).

After the fractions had been collected, they were desalted by gelfiltration in a Sephadex column and were finally freeze-dried.

The ion exchange column was cleaned by washing first with 2.0 M NaCl andthen with 1.0 M NaOH, whereupon the column was ready for the next run.

    ______________________________________                                        Yield of lactoperoxidase after ion exchange:                                                          96.5%.                                                Purity of the collected fraction after elution:                                                       A.sub.412/A280 = 0.84*                                Total yield in the process after                                                                      90%                                                   freeze-drying, calculated as activity:                                        Purity of the freeze-dried                                                                            A.sub.412/A280 = 0.87*                                preparation:                                                                  ______________________________________                                         *0.92 is the maximum quota for 100% purity.                              

The corresponding yield and purity were obtained for the lactoferrin(see FIGS. 3a and 3b).

I claim:
 1. A process for extracting pure fractions of lactoperoxidaseand lactoferrin from milk serum, comprising initially microfiltering themilk serum, then passing it through a bed of a fast flow type strongcation exchanger at a high rate of flow of about 1-1.5 bedvolumes/minute for selective adsorption of lactoperoxidase andlactoferrin, and then successively and selectively eluting thelactoperoxidase with a saline solution having a concentration of0.10-0.4 M at a pH of about 6.5 and the lactoferrin with a salinesolution having a concentration of 0.5-2 M.
 2. The process as claimed inclaim 1, where prior to the elution of lactoperoxidase, the cationexchanger is eluted with a saline solution having a concentration of0.01-0.15 M.
 3. The process as claimed in claim 1 or 2, wherein that thepH of the milk serum is adjusted to 5.9-9.0, preferably about 6.5,before being passed through the cation exchanger.
 4. The process asclaimed claim 1 wherein the microfiltration is carried out in amicrofilter having a pore diameter of 0.10-2 μm.
 5. The process asclaimed claim 1 wherein the saline solutions with eluted lactoperoxidaseand lactoferrin, respectively, are concentrated, desalted andfreeze-dried.
 6. The process as claimed in claim 2, wherein themicrofiltration is carried out in a microfilter having a pore diameterof 0.10-2 μm.
 7. The process as claimed in claim 3, wherein themicrofiltration is carried out in a microfilter having a pore diameterof 0.10-2 μm.
 8. The process as claimed in claim 1, wherein themicrofiltration is carried out in a microfilter having a pore diameterof 0.4-1.5 μm.
 9. The process as claimed in claim 2, wherein themicrofiltration is carried out in a microfilter having a pore diameterof 0.4-1.5 μm.
 10. The process as claimed in claim 3, wherein themicrofiltration is carried out in a microfilter having a bore diameterof 0.4-1.5 μm.
 11. The process as claimed in claim 2, wherein the salinesolutions with eluted lactoperoxidase and lactoferrin, respectively, areconcentrated, desalted and freeze-dried.
 12. The process as claimed inclaim 3, wherein the saline solutions with eluted lactoperoxidase andlactoferrin, respectively, are concentrated, desalted and freeze-dried.13. The process as claimed in claim 4, wherein the saline solutions witheluted lactoperoxidase and lactoferrin, respectively, are concentrated,desalted and freeze-dried.
 14. The process of claim 2 wherein said saltsolution is a solution of an inorganic alkali, alkaline earth orammonium salt.